Previous results have shown that recent thymic emigrants (RTE's) and their immediate descendants in the rat are immature T cells that can be selectively identified and isolated by their expression of the Thy1.1 alloantigen, which is absent from mature T cells. In normal rats, most RTE's resembled medullary thymocyte; in cyclosporin A (CSA)-treated rats, most RTE's resembled cortical thymocyte; in antigen-stimulated (ovalbumin in CFA) rats, most RTE's were antigen-specific, immunoregulatory (IR) T cells; and in autoimmune diabetes-prone BB/W rats, most of the RTE's underwent accelerated apoptosis, resulting in severe depletion of regulatory T cells. We now propose to conduct detailed studies of the phenotypic properties, intrathymic origins, distributions of RTE's and their immediate descendants in normal and antigen-stimulated rats. In addition, we will attempt to determine if RTE's and their immediate descendants in normal rats are immunological competent and/or readily tolerizable; and if IR-RTE's in antigen-stimulated rats are partially activated anergic T cells that secrete non-specific immunoregulatory factors. Purified populations of RTE's and their immediate descendants from normal and antigen-stimulated rats will be isolated by immunomagnetic separation and sorting on the FACS according to their unique Thy1+ TCR alpha/Beta+ RT6-CD45RC-phenotype. Further phenotypic characterization and subset purification of RTE's (including IR-RTE's) and their immediate descendants, will be conducted by 3-color FACS analysis. The origins in thymus cortex or medulla, distribution in peripheral lymphoid tissues, and short-term fate (1-10 days) of RTE's released from the thymus after intrathymic (i.t.) labeling with FITC. The functional capabilities of RTE's their immediate precursors, and their immediate descendants, will be tested in a panel of in vitro assays designed to document lymphocyte activation, lymphokine production, helper/suppressor/cytotoxic activity, and apoptosis. Limiting dilution analyses will be used to estimate the frequencies of reactive cells and to monitor purification. In addition, the relative susceptibility of normal RTE's to the in vitro induction of tolerance with immobilized anti-CD3 will be assessed; and the systems to determine if they ar antigen or IR-RTE's in antigen-stimulated rats will be studied in transwell culture systems to determine if they are antigen or TCR idiotype-specific and if they produce specific or non-specific regulatory factors. To expedite these studies, donors of IR-RTE's will be immunized with antigens known to induce oligoclonal effector T cell response in rats (e.g. MBP, HSP 65kD).